Not applicable
The present invention pertains generally to a method for packing chromatographic columns with a stationary phase and particularly, to a method for packing capillary columns and microchannels.
Chromatography is a method for chemical analysis in which a sample (analyte) consisting of multiple components is introduced into a chromatographic column. As the sample flows through the chromatographic column the individual components of the mixture are separated into distinct bands that are detected near the exit end of the column. The chemical separations are carried out by flowing the sample (analyte) past an immobilized material (the stationary phase) inside the chromatography column. Separation is governed by the dynamic partitioning of the various components of the sample between the analyte and the stationary phase. Control of the separation can be achieved by adjusting the composition of the analyte or the stationary phase or both to influence analyte partitioning.
Conventionally, the stationary phase has been small silica spheres coated with one of a variety of chemical compounds to optimize the chemical separation efficiency. In general, reduction of the chromatography column diameter offers several advantages such as reduced solvent consumption and reduced sample volume requirements. Several chromatography-based analytical methods using miniaturized or capillary columns have been developed. Micromachining techniques have been used to create microchannels 10 to 30 xcexcm wide that can be used for capillary electrophoresis (cf. D. J. Harrison et al., Science, 261, 895, Aug. 13, 1993). However, for packed capillary column methods, such as capillary electrochromatography (CEC) and size exclusion chromatography (SEC), as the column diameters decrease it becomes more difficult to pack the column in a uniform and reproducible way. Irregularities in the uniformity of the stationary phase, both along the length and across the diameter of the column, reduces the efficiency of the chemical separation.
Methods of column packing depend principally on the mechanical strength of the packing, its particle size and particle size distribution, and the diameter of the column to be packed. Conventional column packing methods, such as dry packing, typically used for particles greater than about 20 xcexcm in diameter, are not useful for small capillary columns or microchannels that typically have diameters in the range of tens of microns. For particles between 1 and 20 xcexcm in diameter slurry techniques can be used. In slurry packing the particles that form the bed are suspended as a slurry in an appropriate liquid or liquid mixture. Many liquids or liquid mixtures can be used to prepare the slurry, the principal requirement being that the liquid thoroughly wet the packing particles and provide adequate dispersion of the packing material. The slurry is then pumped into the column. However, as the diameter of the column or channel decreases it becomes necessary to apply higher pressures to force the slurry into and through the column and pressures of 200 to 500 atm are not uncommon. In addition to the obvious hazard of having to work with very high pressures exerted on relatively thin walled tubes, there are other disadvantages to this method of column packing. When the pumping pressure is released at the conclusion of the packing operation the restraining force on the particle bed is partially lost causing an expansion of the particle bed. Then, when the column is once again pressurized heterogeneities or irregularities, such as channels or dead volume, can occur in the particle bed.
Instead of pressure, electro-osmotic flow can be used to carry particles into the capillary from a reservoir of particles suspended in solution. To generate this flow, voltages of from 10 to 30 kV are applied across the capillary. A porous plug or other particle retaining means must be installed at the exit end of the capillary prior to filling to prevent the particles from passing directly through the channel during the filling operation. This method of packing capillary columns suffers the disadvantages of needing very high voltages and a pre-formed porous plug for operation. In general, these methods often do not generate packed beds with optimal uniformity and requires relatively complicated hardware to perform.
Various other approaches have been proposed for introducing a stationary phase into microchannels, in general, and capillaries, in particular, in order avoid the problems associated with pressurized slurry packing. Among these are, coating very small diameter (≈=2-5 xcexcm) microchannel walls, fabricating microstructures, such as xe2x80x9ccolumnsxe2x80x9d or xe2x80x9cpostsxe2x80x9d, within the microchannels itself to serve as the stationary phase, and using xerogels to fill the microchannels.
In prior art processes that employ a packed column for chromatographic analysis, filling a capillary or microchannel with a stationary phase requires that a porous plug or means for retaining the stationary phase within the capillary (while still permitting fluid flow) be put in place prior to the step of filling. This can be a very difficult operation, generally requiring that the material that will composes the porous plug be positioned somehow at the appropriate place in the capillary column. The material is sintered to form a plug that will retain structural integrity as well as a high degree of porosity, while simultaneously fusing the plug to the wall of the capillary.
What is needed is a simple method for filling capillaries and/or microchannels with a stationary phase that eliminates the need for high pressure pumping and forming a retaining means within the capillary column prior to filling with the stationary phase.
It is an object of the present invention to provide a method for packing a stationary phase into a small diameter fluid passageway or flow channel, which can be a microchannel or a capillary, so that the stationary phase is uniformly distributed along both the length and diameter of the flow channel. It is a further object to eliminate the need for forming a porous plug or some other particle retaining means in a flow channel prior to adding the stationary phase. In particular, the present invention is directed toward a method for packing a stationary phase (generally a dielectric material which can or cannot be porous) into flow channels useful for micro-scale high pressure liquid chromatography (HPLC) or capillary electrochromatography (CEC).
The novel method disclosed here is designed to fill flow channels of various materials, whose internal dimensions can be on the order of tens to hundreds of microns, with a uniform distribution of a stationary phase comprising particles of various materials and sizes. The present invention overcomes the well-known and significant deficiencies of prior methods of column packing by causing a suspension of the particles, comprising the stationary phase in an appropriate liquid, to be transported through a flow channel by capillary action. Upon reaching the end of the flow channel the liquid can either evaporate, or flow into a receiving reservoir, leaving the non-volatile stationary phase behind, thereby uniformly filling the flow channel with the desired stationary phase.
The advantages of the method disclosed here are: 1) it eliminates the need for high pressure pumps and fittings and the safety hazards associated therewith for the introduction of a stationary phase into a capillary column; 2) it allows the use of readily available commercial microparticles, that can be coated or uncoated, as the stationary phase for performing chemical separations; 3) different types of particles, particle sizes, and particle size distributions can be packed in sequence, or simultaneously, thereby providing for more complex separations schemes; 4) there is no need for providing means for plugging the flow channel with a porous plug or other particle retaining means to retain the stationary phase prior to adding the stationary phase; and 5) many capillary columns can be filled simultaneously.